Color vision, which differentiates spectral compositions independent of brightness, provides animals, from insects to primates, great power for object recognition and memory registration and retrieval. Using a combination of genetic, histological, electrophysiological and behavioral approaches, we study how the visual system processes chromatic information to generate color percept in Drosophila. Previous primate studies suggest that the color opponent neurons, which exhibit combination-sensitive excitatory and/or inhibitory interaction between two or more photoreceptor types, subserve color vision. Our anatomical study suggests that the Drosophila color-vision circuit in the medulla likely contains such color opponent neurons. We hypothesize that chromatic information is processed in two discrete stages in Drosophila. First, the transmedulla (Tm) neurons serve as color opponent neurons by combining multiple photoreceptor channels and relaying the information to the higher visual center, the lobula. Second, chromatic information is spatially integrated in the lobula, to subserve higher-order color-vision functions, such as color contrast and constancy. Using color preference and color learning assays, we demonstrated that flies innately prefer short wavelengths of light but they can be trained to select specific wavelengths of light by Pavlovian conditioning, indicating that flies, like honeybee and human, have true color vision. Using both light and electron microscope, we determined the synaptic circuits of the photoreceptors and their synaptic target neurons in the medulla. The chromatic photoreceptors, R7 (UV-sensing) and R8 (blue/green-sensing), provide inputs to a subset of first-order interneurons, which might serve as color opponent neurons. The first-order interneurons Tm5a/b/c receive direct synaptic inputs from R7 while Tm9, Tm20 and Tm5c receive inputs from R8. In addition, these Tm neurons receive indirect inputs from R1-6 via L3. These Tm neurons relay spectral information from the medulla to the higher visual center, the lobula. In addition to the direct pathways from photoreceptors to Tm neurons, the amacrine neuron Dm8 receives input from multiple R7s and provides input for Tm5a/b/c. To relate neural connectivity to functions, it is critical to assign components of synaptic machinery to specific connections. To directly probe the usage of neurotransmitters and receptors which determine the polarity and dynamic of signal transmission, we developed a method to profile transcripts in single neurons. We used highly specific promoter-Gal4 constructs to label single types of neurons with GFP and isolated these GFP-labeled neurons from adult fly brains and profiled their gene expression patterns by RT-PCR. Using this method, we determined that the majority of the first-order interneurons in the chromatic circuits express the vesicular glutamate transporter and the Kainate-type of ionotropic glutamate receptors, indicating that they provide and receive fast, sign-conserving glutamate inputs. Our previous studies revealed that the amacrine Dm8 neurons are both required and sufficient for animals' innate spectral preference to UV light while Tm9 neurons are sufficient to drive green phototaxis. RNAi-knock-down of vesicular glutamate transporter significantly reduced UV preference, suggesting that glutamatergic output of Dm8 is critical for its functions. Furthermore, inhibiting the synaptic transmission of its downstream neurons Tm5 also reduced UV preference. Together, we suggest that Tm5 neurons receive excitatory glutamate input from Dm8 neurons to mediate UV phototaxis.